Piwi/Argonaute (Ago) is the only known protein family with function in stem cell self-renewal highly conserved in both animal and plant kingdoms. These proteins also play key roles in germline development and RNAi/miRNA-mediated mechanisms. Our long term goal is to learn molecular mechanisms mediated by the Piwi/Ago proteins in regulating spermatogenesis. This will also contribute to understanding of stem cell division and other developmental processes mediated by this family of proteins. Our strategy has been to use Drosophila as a model to study these proteins in gametogenesis, and then to further what we learn from Drosophila to mammalian and clinical settings. Particularly, in the current funding period, we have demonstrated that mili, a murine member of the gene family, is essential for spermatogonial stem cell division and meiosis, yet miwi, another member, is a key regulator of spermiogenesis. These represent the first two in vivo studies of the piwi/ago gene family in mammalian systems. Moreover, we have shown that the overexpression of a human piwi gene, hiwi, is highly correlated to testicular seminomas. Our specific aim here is to study the molecular mechanisms through which mili and miwi regulate spermatogenesis. Piwi/Ago family proteins are known to negatively regulate gene expression via RNAi, miRNA, and epigenetic pathways. Our latest studies, however, indicate that miwi and mili positively regulate translation and stability of target mRNAs, possibly via a miRNA-mediated mechanism. These findings provide an unparalleled opportunity for us to discover a novel mechanism of gene regulation that is fundamental to reproductive and stem cell biology. Our working hypothesis has three components: First, Mili and Miwi bind to and stabilize target mRNAs required for their target spermatogenic events;second, this function is coupled to translational regulation;third, this function is mediated by miRNAs. To test this hypothesis, we propose to: (1) systematically identify target mRNAs of Mili and Miwi. (2) Characterize the effect of Mili and Miwi binding on the stability of target mRNAs. (3) Determine if Mili and Miwi controls the stability of target mRNAs by regulating their translation. (4) Identify Mili/Miwi-target miRNAs and determine their function in regulating mRNA. Because Piwi/Ago proteins have demonstrated roles in oncogenesis, the proposed studies should bear significant health implications.